The molecular events following inhibition of bacterial peptidoglycan synthesis have not been studied extensively. Previous proteomic studies have revealed that certain proteins are produced in increased amounts upon challenge of Staphylococcus aureus with cell-wall-active antibiotics. In an effort to further those studies, the genes upregulated in their expression in response to cell-wall-active antibiotics have been identified by genome-wide transcriptional profiling using custom-made Affymetrix S. aureus GeneChips. A large number of genes, including ones encoding proteins involved in cell-wall metabolism (including pbpB, murZ, fmt and vraS) and stress responses (including msrA, htrA, psrA and hslO), were upregulated by oxacillin, D-cycloserine or bacitracin. This response may represent the transcriptional signature of a cell-wall stimulon induced in response to cell-wall-active agents. The findings imply that treatment with cell-wall-active antibiotics results in damage to proteins including oxidative damage. Additional genes in a variety of functional categories were upregulated uniquely by each of the three cell-wall-active antibiotics studied. These changes in gene expression can be viewed as an attempt by the organism to defend itself against the antibacterial activities of the agents.